Not applicable.
Not applicable.
Chain termination mutations are those in which a base substitution or frameshift mutation changes a sense codon into one of three stop codons (UAA, UAG, or UGA). Studies of yeast, human genetic disorders, and immunoglobulin family gene expression have identified an RNA surveillance mechanism that minimizes the translation and regulates the RNA stability of nonsense RNAs containing such chain termination mutations. This surveillance mechanism is called xe2x80x9cnonsense-mediated mRNA decayxe2x80x9d (xe2x80x9cNMD,xe2x80x9d see, e.g., Hentze and Kulozik, Cell 96:307-310 (1999); Culbertson, Trends in Genetics 15:74-80 (1999); Li and Wilkinson, Immunity 8:135-141 (1998); and Ruiz-Echevarria et al., TIBS 21-433-438 (1996)). NMD is a post transcriptional mechanism that is operational in both normal cells (i.e., B and T cells) and cells with genetic mutations (i.e., cells with mutations in xcex2-globin, CFTR, and dystrophin).
The NMD machinery discriminates between normal and premature stop codons, and then commits many RNAs with premature stop codons to degradation. In some cases, when the premature stop codon is located near the end of an ORF or in the last exon, the RNA is not subject to NMD and results in production of a truncated polypeptide.
A number of human diseases are caused by nonsense mutations, e.g., p53 associated cancers, retinoblastoma, Duchenne muscular dystrophy, cystic fibrosis, von Willebrand""s disease, thalassemias, neurofibromatosis, Tay-Sachs disease, and hemophilia. In cultured cells having premature stop codons in the CFTR gene, synthesis of full length CFTR was observed when the cells were treated with aminoglycosides (see, e.g., Bedwell et al., Nat. Med. 3:1280-1284 (1997); Howard et al., Nat. Med. 2:467-469 (1996)). Furthermore, in a mouse model for Duchenne muscular dystrophy, gentamycin sulfate was found to suppress translational termination at premature stop codons in the dystrophin gene. These antibiotics mediated misreading and insertion of alternative amino acids at the site of the premature stop codon (see, e.g., Barton-Davis et al., J. Clin Invest. 104:375-381 (1999)). Dystrophin produced in this manner provided protection against contraction-induced damage in the mdx mice.
Compounds that suppress premature translation termination would be a useful treatment for numerous diseases caused by nonsense mutations. Accordingly, high throughput assays for drug discovery related to NMD and inhibition of premature translation termination is desirable. This invention provides these assays, as well as other features which will become apparent upon review.
The present application therefore provides high throughput methods of assaying for compounds that inhibit premature translation termination and nonsense mediated RNA decay in cells.
In one aspect, the present invention provides a method of in vitro screening for compounds that modulate premature translation termination and non-sense-mediated mRNA decay, the method comprising the steps of: (i) incubating a translation assay, the assay comprising an in vitro translation cellular extract; a nucleic acid encoding a polypeptide, wherein the coding sequence for the polypeptide comprises a premature stop codon; and a candidate modulator compound; and (ii) detecting the polypeptide translated from the nucleic acid.
In one embodiment, the cellular extract is from yeast, plants, mammals, or amphibians. In another embodiment, the cellular extract is a eukaryotic reticulocyte lysate, e.g., a rabbit reticulocyte lysate. In another embodiment, the cellular extract is a mammalian tissue culture cell extract, e.g., a HeLa cell S100 extract. In one embodiment, the nucleic acid is an in vitro transcribed RNA.
In another aspect, the present invention provides a method of in vivo screening for compounds that modulate premature translation termination and nonsense-mediated mRNA decay, the method comprising the steps of: (i) expressing in a cell a nucleic acid encoding a polypeptide, wherein the coding sequence for the polypeptide comprises a premature stop codon; (ii) contacting the cell with a candidate modulator compound; and (iii) detecting either the polypeptide translated from the nucleic acid or RNA transcribed from the nucleic acid.
In one embodiment, the nucleic acid comprises a promoter operably linked to a heterologous nucleic acid encoding the polypeptide. In another embodiment, the heterologous nucleic acid encoding the polypeptide comprises an intron and at least two exons comprising coding sequence. In another embodiment, the premature stop codon is located in a last exon. In another embodiment, the heterologous nucleic acid encodes a chimeric polypeptide. In another embodiment, the nucleic acid is an endogenous gene, e.g., an immunoglobulin, xcex1-globin, xcex2-globin, factor VIII, factor IX, vWF, p53, dystrophin, CFTR, Rb, MSH1, MSH2, APC, Wt1, hexosaminidase A, neurofibromin 1, or neurofibromin 2. In another embodiment the cell is adhered to a solid substrate, e.g., a bead, a membrane, and a microtiter plate. In another embodiment, the cell is a human cell or a mouse cell. In another embodiment, the cell is stably transfected with the nucleic acid.
In one embodiment, the nucleic acid encodes an enzyme. In another embodiment, the nucleic acid encodes an immunoglobulin. In another embodiment, the nucleic acid encodes luciferase, green fluorescent protein, red fluorescent protein, phosphatase, peroxidase, kinase, chloramphenicol transferase, or xcex2-galactosidase. In another embodiment, the polypeptide is detected by ELISA, light emission, colorimetric measurements, enzymatic activity, or radioactivity.
In one embodiment, the assay is performed in a well of a microtiter dish, e.g., a microtiter dish having 96 or 384 wells. In another embodiment, the steps of the method are repeated in parallel in a microtiter plate format, wherein between at least about 100 and at least about 6,000 different compounds are tested. In another embodiment, the assay is performed in a high throughput integrated system comprising an automatic pipetting station, a robotic armature, and a robotic controller.
In another aspect, the present invention provides a kit for screening for compounds that modulate translation termination and nonsense-mediated mRNA decay, the kit comprising a nucleic acid encoding a polypeptide, wherein the polypeptide coding sequence comprises a premature stop codon; instructions for practicing a method of screening for compounds that inhibit translation termination at premature stop codons and nonsense mediated RNA decay; and a control compound that inhibits nonsense-mediated RNA decay.
In one embodiment, the control compound is G418 or gentamycin sulfate.